Cellular cofactors affecting hepatitis C virus infection and replication

Abstract

Recently identified hepatitis C virus (HCV) isolates that are infectious in cell culture provide a genetic system to evaluate the significance of virus-host interactions for HCV replication. We have completed a systematic RNAi screen wherein siRNAs were designed that target 62 host genes encoding proteins that physically interact with HCV RNA or proteins or belong to cellular pathways thought to modulate HCV infection. This includes 10 host proteins that we identify in this study to bind HCV NS5A. siRNAs that target 26 of these host genes alter infectious HCV production >3-fold. Included in this set of 26 were siRNAs that target Dicer, a principal component of the RNAi silencing pathway. Contrary to the hypothesis that RNAi is an antiviral pathway in mammals, as has been reported for subgenomic HCV replicons, siRNAs that target Dicer inhibited HCV replication. Furthermore, siRNAs that target several other components of the RNAi pathway also inhibit HCV replication. MicroRNA profiling of human liver, human hepatoma Huh-7.5 cells, and Huh-7.5 cells that harbor replicating HCV demonstrated that miR-122 is the predominant microRNA in each environment. miR-122 has been previously implicated in positively regulating the replication of HCV genotype 1 replicons. We find that 2'-O-methyl antisense oligonucleotide depletion of miR-122 also inhibits HCV genotype 2a replication and infectious virus production. Our data define 26 host genes that modulate HCV infection and indicate that the requirement for functional RNAi for HCV replication is dominant over any antiviral activity this pathway may exert against HCV.

Fig. 1.

Silencing RNAi machinery inhibits HCV replication. siRNAs targeting genes in the RNAi pathway, HCV, or an irrelevant sequence (IRR) were individually introduced into Huh-7.5 cells, which were subsequently infected with HCV. (A) Relative HCV RNA levels were quantified by real-time RT-PCR for HCV and 18S RNA. Values are averages of two sets of triplicates ± SEM. (B) Infectious HCV in cellular supernatants were quantified by limiting dilution and expressed as ID₅₀/ml. (C) Percent inhibition of target RNA levels after siRNA treatment compared with irrelevant treated samples. Values are the levels of target RNA/GAPDH RNA. (D) Cell viability (ATP levels) after siRNA treatment at the time of virus harvesting. ATP levels are normalized to the median of treated samples.

Fig. 2.

Relative miRNA expression profile of liver, Huh-7.5 cell line, and Huh-7.5 with replicating genomic HCV-Con1. miRNAs of human, mouse, and rat were aligned in sequence alignments, and sequence groups were built (specified in SI Table 7). The number of miRNAs in one sequence group is indicated in brackets. The clone count of sequence groups relative to all miRNA clones obtained from liver and Huh7 cell lines is depicted in the specified color code. Tissues were hierarchically clustered based on the miRNA profiles. miRNA expression patterns in the hepatocellular carcinoma cell lines cluster together and separately from liver, as indicated by lines at the top.

Fig. 3.

Interfering with miR-122 inhibits HCV replication. (A) Alignment of miR-122-binding sites in HCV 5′ NTR of genotype 1b Con1 and genotype 2a JFH-1. (B and C) 2′-O-methylated RNAs targeting a random sequence (Rand) or miR-122 (miR-122) were introduced into Huh7.5 cells, infected with HCV, and harvested at the indicated time points, and HCV RNA (B) and infectious virus (C) was quantified. Values are averages of triplicates ± SEM. P value < 0.001.